Two-dimensional electrophoresis with immobilized PH gradients. Current state

Immobilized pit gradients (IPGs) for isoelectric focusing were mtioduu'd in 1982. After ex peri"1 n ci n g initial problems in handling IPGs. a basic protocol o! two-dimensional electrophoresis with IPGs in the first dimension (IPG-Dalti was established in 1988 (Gorg et a!.. Electmphoresis 9, 5:11-046. W}. Since that time, the protocol has not been changed essentially: Isoelectric focusing is performed in individual IPG strips, cast on GelDond PAGfilm. Dry strips are rehydraled to their original thickness with the desired additives, and. afiei IF1 to the steady .state, equilibrated in presence of SOS. DTK, urea, give erol and iodoacetamide, and placed onto the surface of a horizontal ">r on top of a vertical SDS gel. Compared to classical 21) plectrophoresis willi c.trrici arnpholytes. the employment of IPG-Dalt has produced significant improve inenls in 2 I) electroprioretic separation, permitting higher resolution and n producibility which was also demonstrated by inter-laboratoiy comparisons Basic proteins up to pi 12, normally lost by the cathodic drift of carrier am pholyte focusing or separated by NKPHGE with limited reproduc ibility were perfectly separated under equilibrium conditions. These features togethei with the high loading capacity of IPG-Dalt for micropreparative runs have accelcr ated spot identification by microsequencing and mass sped romeuy.